Agarose Electrophoresis (agarose + electrophoresis)

Distribution by Scientific Domains


Selected Abstracts


Separation and recovery of intact gold-virus complex by agarose electrophoresis and electroelution: Application to the purification of cowpea mosaic virus and colloidal gold complex

ELECTROPHORESIS, Issue 17 2004
Carissa M. Soto
Abstract Colloidal gold has been coupled to a mutant cowpea mosaic virus (CPMV), which contains 60 cysteine residues on the surface. A purification process was developed to separate the gold-containing viral nanoblocks (VNBs) from the free gold. Agarose electrophoresis was utilized to separate the mixture followed by electroelution of the desired sample to recover the intact virus. Mobility of Au-VNB and free colloidal gold was facilitated by the addition of thioctic acid (TA). 30% of the gold-containing virus was recovered after electroelution as determined by absorbance measurements. Histogram analysis of transmission electron microscopy (TEM) images demonstrated the efficient separation of gold-containing virus from free gold. TEM and scanning electron microscopy (SEM) images indicated that the virus was recovered intact. Monodisperse spherical particles of nominal size of 45 nm were observed under SEM. [source]


Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

ELECTROPHORESIS, Issue 11 2003
Chad M. Warren
Abstract The electrophoretic separation of high-molecular-weight proteins (>,500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000,4000 kDa) to migrate over 10 cm in a ,13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain (,,220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes. [source]


Computer-assisted 2-D agarose electrophoresis of Haemophilus influenzae type B meningitis vaccines and analysis of polydisperse particle populations in the size range of viruses: A review

ELECTROPHORESIS, Issue 4 2007
Dietmar Tietz Dr.
Abstract When protein,polysaccharide conjugated vaccines were first developed for the immunization of small children against meningitis caused by infection with Haemophilus influenzae type b (Hib), the vaccine preparations varied in immunogenicity. Testing for immunogenicity was time-consuming and alternative analytical procedures for determining vaccine quality were unsatisfactory. For example, due to the very high molecular weight of the vaccine particles, immunogens could only be physically characterized as a fraction in the void volume of Sepharose gel filtration. In search of better analytical methods, a computer-assisted electrophoretic technique for analyzing such vaccines was developed in the period from 1983 to 1995. This new approach made it possible to analyze highly negatively charged particles as large as or larger than intact viruses. 2-D gel patterns were generated that varied depending on the conditions of the particular vaccine preparation and were therefore characteristic of each vaccine sample. Thus, vaccine particle populations with a continuous size variation over a wide range (polydisperse) could be characterized according to size and free mobility (related to particle surface net charge density). These advances are reviewed in this article, since the developed methods are still a promising tool for vaccine quality control and for predicting immunogen effectiveness in the production of vaccines. The technique is potentially beneficial for Hib immunogens and other high-molecular-mass vaccines. Additional biomedical applications for this nondenaturing electrophoretic technique are briefly discussed and detailed information about computational and mathematical procedures and theoretical aspects is provided in the Appendices. [source]


Separation and recovery of intact gold-virus complex by agarose electrophoresis and electroelution: Application to the purification of cowpea mosaic virus and colloidal gold complex

ELECTROPHORESIS, Issue 17 2004
Carissa M. Soto
Abstract Colloidal gold has been coupled to a mutant cowpea mosaic virus (CPMV), which contains 60 cysteine residues on the surface. A purification process was developed to separate the gold-containing viral nanoblocks (VNBs) from the free gold. Agarose electrophoresis was utilized to separate the mixture followed by electroelution of the desired sample to recover the intact virus. Mobility of Au-VNB and free colloidal gold was facilitated by the addition of thioctic acid (TA). 30% of the gold-containing virus was recovered after electroelution as determined by absorbance measurements. Histogram analysis of transmission electron microscopy (TEM) images demonstrated the efficient separation of gold-containing virus from free gold. TEM and scanning electron microscopy (SEM) images indicated that the virus was recovered intact. Monodisperse spherical particles of nominal size of 45 nm were observed under SEM. [source]


Activation of p53 signalling in acetylsalicylic acid-induced apoptosis in OC2 human oral cancer cells

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2003
C.-C. Ho
Abstract Background, Nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid (ASA, aspirin) are well known chemotherapeutic agents of cancers; however, the signalling molecules involved remain unclear. The aim of this study was to investigate the possible existence of a putative p53-dependent pathway underlying the ASA-induced apoptosis in OC2 cells, a human oral cancer cell line. Materials and methods, The methyl tetrazolium (MTT) assay was employed to quantify differences in cell viability. DNA ladder formation on agarose electrophoresis was used as apoptosis assay. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Flow cytometry was used to confirm the effect of ASA on cell cycle. Patterns of changes in expression were scanned and analyzed using the NIH image 156 software (NIH, Bethesda, MD, USA). All the data were analyzed by anova. Results, Acetylsalicylic acid reduced cell viability and presence of internucleosomal DNA fragmentation. In the meanwhile, phosphorylation of p53 at serine 15, accumulation of p53 and increased the expression of its downstream target genes, p21 and Bax induced by ASA. The expression of cyclooxygenase-2 was suppressed. Disruption of p53-murine double minute-2 (MDM2) complex formation resulted in increasing the expression of MDM2 60-kDa cleavage fragment. Inhibited the activation of p42/p44 mitogen-activated protein kinase (MAPK) by PD98059, a specific inhibitor of extracellular regulatory kinase (ERK), significantly decreased cell viability and enhanced the expression of p53 induced by ASA. The result of the cell-cycle analysis showed that ASA and PD98059 induced the cell cycle arrested at the G0/G1 phase and resulted in apoptosis. Conclusion, Nonsteroidal anti-inflammatory drug-inhibited cyclooxygenase is not the only or even the most important mechanism of inhibition. Our study presents evidences that activation of p53 signalling involved in apoptosis induced by ASA. Furthermore, the apoptotic effect was enhanced by blocking the activation of p42/p44 MAPK in response to treatment with ASA, thus indicating a negative role for p42/p44 MAPK. [source]


Alkaline phosphatase isozyme activity in serum from patients with chronic periodontitis

JOURNAL OF PERIODONTAL RESEARCH, Issue 4 2003
P. Gibert
Background:, High alkaline phosphatase activity (ALP) is shown in the periodontal ligament due to the constant renewal of this tissue or pathological circumstances. We have previously shown that the activity level of this enzyme could be reflected at the serum level. Objectives:, Because the local production of ALP in the periodontal ligament is often of the bone-type enzyme, we studied the activity of this isozyme among the other isoforms in the serum of patients with chronic periodontitis in comparison with that of control subjects. Material and methods:, This study included 83 patients (59 with periodontal disease, 24 as control group) and we determined the total seric ALP activity and the percentage of the different isoforms (essentially bone, kidney and intestinal-types) by Ektachem analyser and gel agarose electrophoresis respectively. Conclusions:, By comparisons between the two groups, our results showed a relationship between loss of attachment in periodontal disease and a drop in bone ALP activity in serum. Moreover, these results suggested a gender based difference as well, with lower activity more frequent in women than in men. [source]